Cyclopropanecarboxylic acid, 2-[1-(3,3-dimethylcyclohexyl)ethoxy]-2-methylpropyl ester

Endpoint summary

Description of key information

Skin irritation, in vitro: non-irritating, OECD TG 439, 2008

Skin irritation, in vivo: non-irritating, OECD TG 404, 2004

Eye irritation, in vitro: non-irritating, EPIOCULAR EIT, eq. or similar to OECD TG 492, 2008

Eye irritation, in vivo: non-irritating, OECD TG 405, 2004

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

Endpoint:

skin corrosion: in vitro / ex vivo

Data waiving:

other justification

Justification for data waiving:

an in vitro skin irritation study does not need to be conducted because adequate data from an in vivo skin irritation study are available

other:

Justification for type of information:

JUSTIFICATION FOR DATA WAIVING
In accordance with REACH Regulation (EC) No. 1907/2006 Annex VII, column 2 section 8.1.1 (as amended by Commission Regulation (EU) 2016/863) the in vitro skin corrosion (OECD TG 431) study does not need to be conducted based on the available information allowing a definitive conclusion on the classification of the substance. An available in vivo (eq. or similar to OECD TG 404) skin irritation study is available and data in other endpoints (such as skin sensitisation and/or acute dermal toxicity) indicates that the substance is not skin corrosive and a definitive conclusion on the classification can be made. Furthermore, in accordance with section 1.2 of REACH Regulation (EC) No. 1907/2006 Annex XI the weight of evidence indicates that the substance is not skin corrosive and therefore in vitro testing may be omitted. According to ECHA Guidance on Information Requirements and Chemical Safety Assessment (Chapter R.7a: Endpoint Specific Guidance, R.7.2, July 2017) the study does not need to be conducted.

Reference 2

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

28-05-2008 to 02-06-2008

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Qualifier:

according to guideline

Guideline:

other: ECVAM international validation study on in vitro tests for acute skin irritation: Report on the validity of the EPISKIN and EpiDerm assays and on the Skin Integrity Function Test

Version / remarks:

Reference: Altern Lab Anim. 2007 Dec; 35 (6): 559-601

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)

Deviations:

no

Qualifier:

equivalent or similar to guideline

Guideline:

EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

inspected: September 2006 ; signature: January 2007

Test system:

human skin model

Remarks:

EPISKIN TM Small Model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

foreskin from a single donor

Vehicle:

unchanged (no vehicle)

Details on test system:

EpiSkin RHE Model (Lot no.: 08-EKIN-020). The principle of the assay is based on the measurement of cytotoxicity in reconstructed human epidermal cultures following topical exposure to the test item by means of the colorimetric MTT reduction assay. Cell viability is measured by enzymatic reduction of the yellow MTT tetrazolium salt. to a blue formazan salt (within the mitochondria of viable cells) in the test item treated tissues relative to the negative controls. The EPISKIN model is a three-dimensional reconstructed human epidermis model consisting of adult human-derived epidermal keratinocytes seeded on a dermal substitute consisting of a collagen type I matrix coated with type IV collagen. A highly differentiated and stratified epidermis model is obtained after a 13-Day culture period comprising of the main basal, supra basal, spinous and granular layers and a functional stratum corneum. Pre-test checks for Direct MTT reduction and Colour Interference were completed.

Preincubation:
After approx. 24 hours incubation of the EpiSkin-SM tissues, they were treated with the test item. Under sterile conditions using sterile forceps, the inserts were transferred into 12-well plates containing the pre-warmed (37 ± 1 °C) maintenance medium.

Application of test item and rinsing:
The negative and positive control, and the test item were added into the insert atop the concerning EPISKIN-SMTM triplicate tissues. Solid: Additionally, the test item tissues were wetted with 15 μL of deionised water. The 12-well plates were placed into the incubator for 15 ± 1 min at 37 ± 1°C, 5 ± 0.5% CO2. After the end of the treatment interval the inserts were removed immediately from the 12-well plate. Using a wash bottle the tissues were gently rinsed with PBS to remove any residual test material. Excess PBS was removed by gently shaking the inserts and blotting the bottom with blotting paper. The inserts were placed in the plates with 2 mL maintenance medium. Tissues were incubated for 42 ± 1 hours at 37 ± 1°C, 5 ± 0.5% CO2.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 15 µL
- Concentration (if solution): undiluted

Duration of treatment / exposure:

Tissues were treated with the test material for 15 minutes and then washed with PBS to remove residual test material.
Positive control: SLS (5%) solution, 15 μL for 15 ± 1 min.
Negative control: Deionised water, 15 μL for 15 ± 1 min.

Duration of post-treatment incubation (if applicable):

Subsequently the skin tissues were incubated for 42 hours at 37°C.

Number of replicates:

Triplicate; treatment and concurrent negative control and positive control groups

Details on study design:

TEST SITE
- Area of exposure: 15 μl of the undiluted test substance was added topically into 12-well plates on top of the skin tissues.

REMOVAL OF TEST SUBSTANCE
- Washing (if done): yes
- Time after start of exposure: 15 minutes

SCORING SYSTEM: Skin irritation potential of the test substance was classified according to remaining cell viability following exposure of the test substance.

Irritation / corrosion parameter:

% tissue viability

Remarks:

mean tissue viability

Value:

92.6

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other:

Remarks:

Basis: mean. Time point: 15 minute exposure. Reversibility: no data. Remarks: n=3 ; SD = 8% ; Score in terms of percentage of negative control.

Other effects / acceptance of results:

- OTHER EFFECTS:
- Visible damage on test system: None reported.
- Direct-MTT reduction: Screened prior to test. Not applicable.
- Colour interference with MTT: Not screened.

DEMONSTRATION OF TECHNICAL PROFICIENCY:

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes.
- Acceptance criteria met for positive control: Yes.
- Acceptance criteria met for variability between replicate measurements: Yes.
- Range of historical values if different from the ones specified in the test guideline: Not applicable.

Interpretation of results:

GHS criteria not met

Remarks:

Criteria used for interpretation of results: EU

Conclusions:

Under the conditions of this in vitro study, the test item is not considered to be irritating to skin.

Executive summary:

The study was performed according to the method described in the ECVAM international validation study on in vitro tests for acute skin irritation: Report on the validity of the EPISKIN and EpiDerm assays and on the Skin Integrity Function Test (Aitern Lab Anim. 2007 Dec; 35 (6): 559-601) in accordance with GLP and/or methodology equivalent to later OECD TG 439 and EU Method B.46 guidelines, to assess the skin irritation potential of the test substance using a human three dimensional epidermal model (EPISKIN Small Model). Triplicate tissues were treated with the test item for an exposure period of 15 minutes. At the end of the exposure period each tissue was rinsed before incubating for 42 hours. At the end of the post-exposure incubation period each tissue was taken for MTT-loading. After MTT-loading a total biopsy of each epidermis was made and placed into micro tubes containing acidified isopropanol for extraction of formazan crystals out of the MTT-loaded tissues. At the end of the formazan extraction period each tube was mixed thoroughly and duplicate 200 μL samples were transferred to the appropriate wells of a pre-labelled 96 -well plate. The optical density was measured at 570 nm. Data are presented in the form of percentage viability (MTT reduction in the test item treated tissues relative to negative control tissues). The relative mean viability of the test item treated tissues was 92.6% after the 15 -Minute exposure period and 42 -Hours post-exposure incubation period. All assay acceptability criteria were considered to be met. Under the conditions of this study, the test item is considered to be not irritating to the skin.

Reference 3

Endpoint:

skin irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

03-06-2004 to 19-08-2004

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study performed under GLP. All relevant validity criteria were met.

Qualifier:

according to guideline

Guideline:

OECD Guideline 404 (Acute Dermal Irritation / Corrosion)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.4 (Acute Toxicity: Dermal Irritation / Corrosion)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

inspected: November 2002 ; signature: March 2003

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Recognised supplier
- Age at study initiation: 11 - 12 weeks (male), 10 - 11 weeks (female)
- Weight at study initiation: 2.150 - 2.474 kg
- Housing: Individually housed in stainless steel cages equipped with feed hoppers and water bowls; wood blocks and haysticks provided for gnawing (cage enrichment).
- Diet: Pelleted standard rabbit maintenance diet, ad libitum. Details in the full study report.
- Water: Community tap water, ad libitum.
- Acclimation period: Time not specified

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 17 - 23 (values outside this range may have occasionally occurred during cleaning)
- Humidity (%): 30 - 70 (values outside this range may have occasionally occurred during cleaning)
- Air changes (per hr): 10 - 15
- Photoperiod: 12 hours light with music / 12 hours dark

Type of coverage:

semiocclusive

Preparation of test site:

clipped

Vehicle:

unchanged (no vehicle)

Controls:

no

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.5 mL
- Concentration (if solution): Test material was used as supplied.

Duration of treatment / exposure:

4 hours

Observation period:

1, 24, 48 and 72 hours after treatment.

Number of animals:

3

Details on study design:

TEST SITE
- Area of exposure: dorsal
- Type of wrap if used: semi-occlusive (4 cm x 4 cm surgical gauze patch secured with surgical adhesive tape)

REMOVAL OF TEST SUBSTANCE
- Washing (if done): Yes, any residual test item removed by irrigation with lukewarm tap water.
- Time after start of exposure: 4 hours

SCORING SYSTEM:
Erythema and Eschar Formation
No erythema _________________________________________________________________________0
Very slight erythema (barely perceptible) ________________________________________________1
Well-defined erythema ________________________________________________________________2
Moderate to severe erythema __________________________________________________________3
Severe erythema (beet redness) to slight eschar formation (injuries in depth) ________________4

Oedema Formation
No oedema __________________________________________________________________________0
Very slight oedema (barely perceptible) _________________________________________________1
Slight oedema (edges of area well-defined by definite raising) _____________________________2
Moderate oedema (raised approximately 1 millimetre) ____________________________________3
Severe oedema (raised more than 1 millimetre and extending beyond the area of exposure) ___4
Any other skin reactions and clinical signs of toxicity, if present, were also recorded.

Irritation parameter:

erythema score

Basis:

animal #1

Time point:

other: 1h

Score:

2

Max. score:

4

Reversibility:

fully reversible within: 48 hours

Irritation parameter:

erythema score

Basis:

animal #2

Time point:

other: 1h

Score:

1

Max. score:

4

Reversibility:

fully reversible within: 48 hours

Irritation parameter:

erythema score

Basis:

animal #3

Time point:

other: 1 h

Score:

2

Max. score:

4

Reversibility:

fully reversible within: 72 hours

Irritation parameter:

erythema score

Basis:

animal #1

Time point:

24 h

Score:

1

Max. score:

4

Reversibility:

fully reversible within: 48 hours

Irritation parameter:

erythema score

Basis:

animal #2

Time point:

24 h

Score:

1

Max. score:

4

Reversibility:

fully reversible within: 48 hours

Irritation parameter:

erythema score

Basis:

animal #3

Time point:

24 h

Score:

2

Max. score:

4

Reversibility:

fully reversible within: 72 hours

Irritation parameter:

erythema score

Basis:

animal #1

Time point:

48 h

Score:

0

Max. score:

4

Irritation parameter:

erythema score

Basis:

animal #2

Time point:

48 h

Score:

0

Max. score:

4

Irritation parameter:

erythema score

Basis:

animal #3

Time point:

48 h

Score:

1

Max. score:

4

Reversibility:

fully reversible within: 72 hours

Irritation parameter:

erythema score

Basis:

animal #1

Time point:

72 h

Score:

0

Max. score:

4

Irritation parameter:

erythema score

Basis:

animal #2

Time point:

72 h

Score:

0

Max. score:

4

Irritation parameter:

erythema score

Basis:

animal #3

Time point:

72 h

Score:

0

Max. score:

4

Irritation parameter:

edema score

Basis:

animal #1

Time point:

other: 1 h

Score:

1

Max. score:

4

Reversibility:

fully reversible within: 24 h

Irritation parameter:

edema score

Basis:

animal #2

Time point:

other: 1 h

Score:

0

Max. score:

4

Irritation parameter:

edema score

Basis:

animal #3

Time point:

other: 1 h

Score:

0

Max. score:

4

Irritation parameter:

edema score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0

Max. score:

4

Irritation parameter:

edema score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0

Max. score:

4

Irritation parameter:

edema score

Basis:

animal #3

Time point:

24/48/72 h

Score:

0

Max. score:

4

Irritant / corrosive response data:

- Erythema: Well-defined erythema (score = 2) in 2 sites, and slight erythema (score = 1) in 1 site, 1 hour after treatment. Partial reversal of effects (score = 1) in one site by 24 hours, and in the other sites (scores = 1 and 0) by 48 hours. Complete reversal by 72 hours.
- Edema: Slight edema (score = 1) observed in 1 site, 1 hour after treatment, with effects fully-reversed within 24 hours.
- Reversibility of effects: All effects reversed within 72 hours.

Other effects:

All animals gained body weight during the study period. The body weights of all rabbits were considered to be within the normal range of variablity. There was no clinical signs of toxicity reported during the duration of the test.

Table 1. Individual skin reactions

Skin Reaction	Observation Time (following patch removal)	Individual Scores
		Number and Sex
		#1 (male)	#2 (female)	#3 (female)
Erythema/Eschar Formation	1 Hour	2	1	2
	24 Hours	1	1	2
	48 Hours	0	0	1
	72 Hours	0	0	0
Edema Formation	1 Hour	1	0	0
	24 Hours	0	0	0
	48 Hours	0	0	0
	72 Hours	0	0	0

 

Mean scores (N=3) per organism at 24, 48 and 72h:

Erythemea/Escar Formation:

1: 0.33

2: 0.33

3: 1.00

Edema Formation:

1: 0.00

2: 0.00

3: 0.00

Interpretation of results:

GHS criteria not met

Remarks:

Criteria used for interpretation of results: EU

Conclusions:

Under the conditions of this study the test item is not considered to be irritating.

Executive summary:

The study was performed to OECD TG 404 and EU Method B.4 under GLP, to assess the primary skin irritancy potential of the test item in New Zealand White rabbits. Following single 4 -Hour, semi-occluded applications to the intact rabbit skin. 0.5 mL of the test item was introduced under a 4 cm x 4 cm cotton gauze patch and placed in position on the clipped skin to assess the irritancy potential of the test item. The patch was secured in position with a strip of surgical adhesive tape. After 4 hours of exposure to the test item, the patches were removed and individual dose sites were scored at approximately 1, 24, 48, and 72 hours. A single 4 -Hour, semi occluded application of the test item to the intact skin of two rabbits produced well defined erythema at two treated skin sites 1 hour after patch removal, which was partially reversed in one site within 24 hours and the other site within 48 hours. Slight erythema was detected 1 hour after patch removal in 1 site, which was fully reversed within 48 hours. All erythema was fully reversed within 72 hours. Slight edema was also observed in 1 site, 1 hour after patch removal, which was fully reversed within 24 hours. No corrosive effects were noted. Mean scores for following grading at 24, 48 and 72 hours were 0.33, 0.33 and 1.00 in erythema and eschar, and 0.00 in edema scoring criteria. Under the conditions of the study, the test item is not considered to be a skin irritant.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Reference 1

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

12-06-2008 to 13-06-2008

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 492 (Reconstructed Human Cornea-like Epithelium (RhCE) Test Method for Identifying Chemicals Not Requiring Classification and Labelling for Eye Irritation or Serious Eye Damage)

Version / remarks:

MatTek Corporation EpiOcular™ Eye Irritation Test (OCL-200-EIT)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

inspected: September 2006 ; signature: January 2007

Species:

other: human-derived epidermal keratinocyte tissues

Strain:

not specified

Details on test animals or tissues and environmental conditions:

- Justification of the test method and considerations regarding applicability: The test method is indicated as a validated test method recommended by OECD and/or recognised internationally for the sequential testing strategy for the assessment of ocular irritation.
- Description of the cell system used, incl. certificate of authenticity and the mycoplasma status of the cell live: EpiOcular™ (OCL-200-EIT MatTek Corporation, Lot: 10231 Kit F).

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 50 µL of the test item
- Concentration (if solution): undiluted

VEHICLE
- Amount(s) applied (volume or weight with unit): Not applicable.
- Concentration (if solution): Not applicable.
- Lot/batch no. (if required): Not applicable.
- Purity: Not applicable.

Duration of treatment / exposure:

Negative control: 60 minutes at 37 ± 1°C
Positive control: 15 and 45 minutes at 37 ± 1°C
Test item: 3, 30 and 60 minutes at 37 ± 1°C

Duration of post- treatment incubation (in vitro):

10 to 20 min.

Number of animals or in vitro replicates:

Two tissues were used for each treatment group (test item, negative control and positive control).

Details on study design:

- Details of the test procedure used: See below.
- RhCE tissue construct used, including batch number: EpiOcular™ (OCL-200-EIT MatTek Corporation, Lot: 10231 Kit F).
- Doses of test chemical and control substances used: 100 µL test item / 100 µL deionised water (negative control) / 100 µL 0.3% Triton X-100 (positive control)
- Duration and temperature of exposure, post-exposure immersion and post-exposure incubation periods (where applicable): After the incubation, the tissues were pre-wetted with 300 µL of assay medium. The tissues were incubated at standard culture conditions (37 ± 1.5 °C, 5 ± 0.5% CO2) until use. After the pre-treatment, the test and control items were tested by applying 100 µL topically on the EpiOcular tissues. The tissues were incubated at standard culture conditions for 60 minutes (negative control), 15 and 45 minutes (positive control) and 3, 30 and 60 minutes (test item). At the end of the treatment time, the test item was removed by extensively rinsing the tissues with PBS (brought to room temperature). After rinsing, the tissues were immediately transferred to and immersed in 5 mL of previously warmed Assay Medium (room temperature) in pre-labelled 6-well plates for a 10-20 minute immersion incubation (post-soak) at room temperature. This incubation in Assay Medium was intended to remove any test item absorbed into the tissue. At the end of the post-soak immersion, each insert was removed from the Assay Medium, the medium was aspirated off the tissue, and the wells were triply rinsed with PBS. The inserts were immersed into extractant solution (2 mL isopropanol) for formazan salt extraction, sealed, and incubated for 18 hours without agitation at room temperature. After the extraction period, the inserts were pierced with an injection needle to allow the extract to run into the well from which the insert was taken. Afterwards the insert was discarded. The well plates were placed on a shaker for 15 minutes until the solution was homogeneous in colour. 3 x 200 µL aliquots of the formazan blue solution were transferred to a well plate and OD was read in a microplate reader at 570 nm without reference filter. Mean values (n=3) were calculated.
- Justification for the use of a different negative control than ultrapure H2O (if applicable): Not applicable.
- Justification for the use of a different positive control than neat methyl acetate (if applicable): Not specified.
- Description of any modifications to the test procedure: Not specified.
- Indication of controls used for direct MTT-reducers and/or colouring test chemicals (if applicable): The test item did not directly-reduce MTT or was colour inference in pre-checks conducted prior to the exposure.
- Number of tissue replicates used per test chemical and controls (positive control, negative control, NSMTT, NSCliving and NSCkilled, if applicable): Duplicate.
- Wavelength and band pass (if applicable) used for quantifying MTT formazan, and linearity range of measuring device (e.g. spectrophotometer): 570 nm
- Description of the method used to quantify MTT formazan: Versamax® Molecular Devices, 85737 Ismaning, Germany – spectrophotometrically.
- Description of the qualification of the HPLC/UPLC-spectrophotometry system (if applicable): Not applicable.
- Description of evaluation criteria used including the justification for the selection of the cut-off point for the prediction model: Not specified.
- Reference to historical positive and negative control results demonstrating suitable run acceptance criteria: Not specified.
- Complete supporting information for the specific RhCE tissue construct used: Attached to the full study report.
- Reference to historical data of the RhCE tissue construct: Full historic control data is attached to the full study report.
- Demonstration of proficiency in performing the test method before routine use by testing of the proficiency chemicals: The test assay is validated at the test laboratory and certification of technical proficiency from the model supplier is attached to the full study report.
- Positive and negative control means and acceptance ranges based on historical data: See above.
- Acceptable variability between tissue replicates for positive and negative controls: Yes, < 6.2 % for positive control, negative control and test item replicates.
- Acceptable variability between tissue replicates for the test chemical: Yes.

Irritation parameter:

other: Mean tissue viability (% of negative control)

Remarks:

n=2

Run / experiment:

mean - test item (60 min)

Value:

114

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Irritation parameter:

other: Mean tissue viability (% of negative control)

Remarks:

n=2

Run / experiment:

mean - positive control (45 min)

Value:

22.2

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Other effects / acceptance of results:

The ET50-value of the positive control was calculated to be 14.6 min. An ET50-value could not be calculated for the test item because viability was not reduced, where:
ET50 = a - ((a - b)(c - 50)) / (c - d)
where:
a = max. measured time in min with the% of negative control > 50%
b = min. measured time in min with the% of negative control < 50%
c = relative absorbance at time a in %
d = relative absorbance at time b in %

The lower the ET 50 value, the higher the eye irritant/cytotoxic potential of the test item. For classification purposes, an ET50-value greater than 60 is a non/minimal irritant; a value of 31-60 denotes a mild irritant; 3-30 denotes a moderate irritant and <3 denotes a severe/extreme irritant.

Based on the inability to calculate an ET50, the test item is not predicted to be an eye irritant.

OTHER EFFECTS:
- Visible damage on test system: None.

DEMONSTRATION OF TECHNICAL PROFICIENCY: The test assay is validated at the test laboratory and certification of technical proficiency from the model supplier is attached to the full study report.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes.
- Acceptance criteria met for positive control: Yes.
- Range of historical values if different from the ones specified in the test guideline: See 'details on study design' for further information.

Table 1.0 : Mean absorption and Mean Tissue Viability in the EPIOCULAR Test

Dose Group	Treatment Interval	Mean Absorbance of 2 Tissues	Rel. Absorbance (% of Negative Control)
Negative Control	60 min	1.7315	100.0
Positive Control	15 min	0.8454	48.8
	45 min	0.3840	22.2
Test Item	3 min	1.7749	102.5
	30 min	2.0066	115.9
	60 min	1.9739	114.0

Acceptability of the Assay

a) The absolute mean OD570 of the two tissues of the negative control should reasonably be > 0.8 and < 2.5.

b) The mean relative tissue viability of the positive control should be < 50% relative to the negative control.

c) The difference between the % tissue viabilities of the two identically treated replicates should be < 20%.

All assay acceptability criteria were met.

Interpretation of results:

GHS criteria not met

Remarks:

Criteria used for interpretation of results: EU

Conclusions:

Under the conditions of this study, the test item is not considered to be an eye irritant.

Executive summary:

The study was performed to assess the eye irritancy potential of the test item using the EpiOcular EIT model under OECD TG 492 guideline in accordance with GLP. The test item was found to not directly reduce MTT or colour interfere in pre-test checks. The test item was topically applied for 3, 30 and 60 minutes. After exposure the cornea epithelial construct was thoroughly rinsed to remove the test item and transferred to fresh medium for an immersion incubation. The tissues were transferred to fresh medium and incubated under standard culture conditions, prior to determination of the cytotoxic (irritancy) effect. The relative mean tissue viability obtained after treatment with the test item compared to the negative control tissues was 114% (60 min). The positive control had a mean cell viability of 22.2% after 45 minutes treatment. The absolute mean OD570 (optical density at 570 nm) of the negative control tissues was within the acceptability criteria. The difference between the percentages of viability of two tissues treated identically was less than 20%, indicating that the test system functioned properly. Under the conditions of this study, since the test item relative mean viability was > 60% the test item is not predicted to be eye irritant.

Reference 2

Endpoint:

eye irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2-06-2004 to 28-09-2004

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study performed under GLP. All relevant validity criteria were met.

Qualifier:

according to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.5 (Acute Toxicity: Eye Irritation / Corrosion)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

inspected: November 2002 ; signature: March 2003

Species:

rabbit

Strain:

New Zealand White

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
- Source: Recognised supplier
- Age at study initiation: 14 weeks (male), 12 weeks (females)
- Weight at study initiation: 2.017 - 2.239 kg
- Housing: individually housed in suspended metal (stainless steel) cages; with environment enrichment wood blocks and hay sticks for gnawing (cage enrichment).
- Diet: Pelleted standard rabbit maintenance diet, ad libitum. Details in the full study report.
- Water: Community tap water, ad libitum
- Acclimation period: Time not specified

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 17 - 23 (deviations possible during room cleaning).
- Humidity (%): 30 - 70 (deviations possible during room cleaning).
- Air changes (per hr): 10-15
- Photoperiod (hrs dark / hrs light): 12 hour light with music / 12 hour dark

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent no treatment

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.1 mL (per animal)
- Concentration (if solution): undiluted

Duration of treatment / exposure:

A volume of 0.1 mL of the test material, was placed into the conjunctival sac of the left eye, formed by gently pulling the lower lid away from the eyeball. The upper and lower eyelids were held together for about one second immediately after treatment, to prevent loss of the test material, and then released. The right eye remained untreated and was used for control purposes.

Observation period (in vivo):

Ocular assessment was conducted at approximately 1, 24, 48 and 72 hours after instillation of the test substance, according to numerical evaluation.

Number of animals or in vitro replicates:

3 (1 male and 2 female).

Details on study design:

REMOVAL OF TEST SUBSTANCE
- Washing (if done): none

SCORING SYSTEM:
The ocular reaction was assessed according to the numerical scoring system listed in the EEC Commission Directive 92/69/EEC, July 31, 1992 at approximately 1, 24, 48 and 72 hours after instillation. Scoring system consistent with Draize scoring system. When present, corrosion and/or staining of conjunctivae, sclerae and cornea by the test item were recorded and reported.

TOOL USED TO ASSESS SCORE: Eye examinations were made with a Varta Cliptrix diagnostic-lamp.

Irritation parameter:

cornea opacity score

Basis:

mean

Time point:

24/48/72 h

Score:

0

Max. score:

4

Remarks on result:

other: mean; n=3

Irritation parameter:

iris score

Basis:

mean

Time point:

24/48/72 h

Score:

0

Max. score:

2

Remarks on result:

other: mean; n=3

Irritation parameter:

conjunctivae score

Basis:

mean

Time point:

24/48/72 h

Score:

0

Max. score:

3

Remarks on result:

other: mean; n=3

Irritation parameter:

chemosis score

Basis:

mean

Time point:

24/48/72 h

Score:

0

Max. score:

4

Remarks on result:

other: mean; n=3

Irritant / corrosive response data:

No corneal effects were noted. Slight reddening of the conjunctivae of treated eyes was observed 1 hour after treatment, although fully resolved by the 24-hour observation. Slight chemosis of the conjunctivae was observed in one animal 1 hour after treatment, although fully resolved by the 24-hour observation. Slight reddening of the sclerae was present in all animals 1 hour after treatment, although fully resolved by the 24-hour observation. No abnormal findings were observed in the treated eye of any animal 24 hours after treatment.

Other effects:

- Lesions and clinical observations: None reported.
- Ophthalmoscopic findings: None reported.
- Histopathological findings: Not applicable.
- Effects of rinsing or washing: Not applicable.
- Other observations: Not applicable.

Table 1. Individual scores and mean scores for 24, 48 and 72 hours

Organism number	1					2					3
Time After Treatment	1 Hour	24 Hours	48 Hours	72 Hours	7 Days	1 Hour	24 Hours	48 Hours	72 Hours	7 Days	1 Hour	24 Hours	48 Hours	72 Hours	7 Days
CORNEA
Degree of Opacity	0	0	0	0	-	0	0	0	0	-	0	0	0	0	-
Mean (24 – 72 h)				0	-				0	-				0	-
Area of Cornea Involved	0	0	0	0	-	0	0	0	0	-	0	0	0	0	-
IRIS	0	0	0	0	-	0	0	0	0	-	0	0	0	0	-
Mean (24 – 72 h)				0	-				0	-				0	-
CONJUNCTIVAE					-					-					-
Redness	1	0	0	0	-	1	0	0	0	-	1	0	0	0	-
Mean (24 – 72 h)				0	-				0	-				0	-
Chemosis	0	0	0	0	-	1	0	0	0	-	0	0	0	0	-
Mean (24 – 72 h)				0	-				0	-				0	-

Interpretation of results:

GHS criteria not met

Remarks:

Criteria used for interpretation of results: EU

Conclusions:

Under the conditions of this study the test item is not irritating to the eye.

Executive summary:

The study was performed to OECD TG 405 and EU Method B.5 under GLP to assess the irritancy potential of the test item to the eye following a single application in the New Zealand White rabbit. A volume of 0.1 ml of the test material was placed into the conjunctival sac of one eye of three rabbits. The other eye remained untreated and was used for control purposes. Assessment of ocular damage/irritation was made approximately 1 hour and 24, 48 and 72 hours following treatment. A single application of the test item to the non-irrigated eye of three rabbits produced no corneal effects. Slight redness of the conjunctivae and sclera was observed in all treated eyes 1 hour after treatment, although this was absent at the 24 hour observation and onwards. Slight chemosis was observed in one treated eye 1 hour after treatment, which was absent at the 24 hour examination and subsequent examinations. All treated eyes appeared normal at the 72 hours observation. Under the conditions of this study, the test item is not considered to be irritating to the eye.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Additional information

Skin Irritation:

Key study: in vitro, eq. or similar to OECD TG 439, 2008 : The study was performed according to the method described in the ECVAM international validation study on in vitro tests for acute skin irritation: Report on the validity of the EPISKIN and EpiDerm assays and on the Skin Integrity Function Test (Aitern Lab Anim. 2007 Dec; 35 (6): 559-601) in accordance with GLP and/or methodology equivalent to later OECD TG 439 and EU Method B.46 guidelines, to assess the skin irritation potential of the test substance using a human three dimensional epidermal model (EPISKIN Small Model). Triplicate tissues were treated with the test item for an exposure period of 15 minutes. At the end of the exposure period each tissue was rinsed before incubating for 42 hours. At the end of the post-exposure incubation period each tissue was taken for MTT-loading. After MTT-loading a total biopsy of each epidermis was made and placed into micro tubes containing acidified isopropanol for extraction of formazan crystals out of the MTT-loaded tissues. At the end of the formazan extraction period each tube was mixed thoroughly and duplicate 200 μL samples were transferred to the appropriate wells of a pre-labelled 96 -well plate. The optical density was measured at 570 nm. Data are presented in the form of percentage viability (MTT reduction in the test item treated tissues relative to negative control tissues). The relative mean viability of the test item treated tissues was 92.6% after the 15 -Minute exposure period and 42 -Hours post-exposure incubation period. All assay acceptability criteria were considered to be met. Under the conditions of this study, the test item is considered to be not irritating to the skin.

Key study: in vivo, OECD TG 404, 2004 : The study was performed to OECD TG 404 and EU Method B.4 under GLP, to assess the primary skin irritancy potential of the test item in New Zealand White rabbits. Following single 4 -Hour, semi-occluded applications to the intact rabbit skin. 0.5 mL of the test item was introduced under a 4 cm x 4 cm cotton gauze patch and placed in position on the clipped skin to assess the irritancy potential of the test item. The patch was secured in position with a strip of surgical adhesive tape. After 4 hours of exposure to the test item, the patches were removed and individual dose sites were scored at approximately 1, 24, 48, and 72 hours. A single 4 -Hour, semi occluded application of the test item to the intact skin of two rabbits produced well defined erythema at two treated skin sites 1 hour after patch removal, which was partially reversed in one site within 24 hours and the other site within 48 hours. Slight erythema was detected 1 hour after patch removal in 1 site, which was fully reversed within 48 hours. All erythema was fully reversed within 72 hours. Slight edema was also observed in 1 site, 1 hour after patch removal, which was fully reversed within 24 hours. No corrosive effects were noted. Mean scores for following grading at 24, 48 and 72 hours were 0.33, 0.33 and 1.00 in erythema and eschar, and 0.00 in edema scoring criteria. Under the conditions of the study, the test item is not considered to be a skin irritant.

Eye Irritation:

Key study: In vitro, eq. or similar to OECD TG 492, 2008 : The study was performed to assess the eye irritancy potential of the test item using the EpiOcular EIT model under OECD TG 492 guideline in accordance with GLP. The test item was found to not directly reduce MTT or colour interfere in pre-test checks. The test item was topically applied for 3, 30 and 60 minutes. After exposure the cornea epithelial construct was thoroughly rinsed to remove the test item and transferred to fresh medium for an immersion incubation. The tissues were transferred to fresh medium and incubated under standard culture conditions, prior to determination of the cytotoxic (irritancy) effect. The relative mean tissue viability obtained after treatment with the test item compared to the negative control tissues was 114% (60 min). The positive control had a mean cell viability of 22.2% after 45 minutes treatment. The absolute mean OD570 (optical density at 570 nm) of the negative control tissues was within the acceptability criteria. The difference between the percentages of viability of two tissues treated identically was less than 20%, indicating that the test system functioned properly. Under the conditions of this study, since the test item relative mean viability was > 60% the test item is not predicted to be eye irritant.

Key study : In vivo, OECD TG 405, 2004 : The study was performed to OECD TG 405 and EU Method B.5 under GLP to assess the irritancy potential of the test item to the eye following a single application in the New Zealand White rabbit. A volume of 0.1 ml of the test material was placed into the conjunctival sac of one eye of three rabbits. The other eye remained untreated and was used for control purposes. Assessment of ocular damage/irritation was made approximately 1 hour and 24, 48 and 72 hours following treatment. A single application of the test item to the non-irrigated eye of three rabbits produced no corneal effects. Slight redness of the conjunctivae and sclera was observed in all treated eyes 1 hour after treatment, although this was absent at the 24 hour observation and onwards. Slight chemosis was observed in one treated eye 1 hour after treatment, which was absent at the 24 hour examination and subsequent examinations. All treated eyes appeared normal at the 72 hours observation. Under the conditions of this study, the test item is not considered to be irritating to the eye.

Respiratory Irritation:

Key study: in vivo, OECD TG 403, 2015 : The study was performed according to OECD TG 403, EU Method B.2, US EPA OPPTS 870.1300 and Japanese JMAFF guidelines in accordance with GLP to assess the acute inhalation toxicity of the test item. A single group of ten Wistar: Crl:WI(Han) strain rats (five males and five females) were exposed to an aerosol atmosphere of the test item. The groups were exposed for four hours using a nose only exposure system, followed by a fifteen day observation period. The mean achieved atmosphere concentrations were as follows: 5.1 ± 0.1 mg/L based on a nominal concentration of 7.8 mg/L. The characteristics of the achieved atmosphere where Mean Mass Median Diameter (particle size): > 2.3 μm and < 2.4 μm with geometric Standard Deviation > 1.9 and < 2.2. There were no mortalities in the 5.1 mg/L mean achieved atmosphere concentration. Slow breathing was noted during exposure. After exposure, no clinical signs were observed among the males, whereas hunched posture was observed on days 2 and 3 only for one female. The affected female had recovered from the signs between Days 3 and 4. Body weight loss was noted in some male/females during the first two days post-exposure. All males/females regained weight during the remainder of the observation period. No abnormalities were found at macroscopic post-mortem examination. Under the conditions of this study, the inhalation 4h-LC50 (male/female) was considered to be > 5.1 mg/L within the Wistar: Crl:WI(Han) rat. Under the conditions of this study, there were no indications of respiratory irritation.

 

References:

1. OECD TG 403 (2009)

2. OECD 39 (2009)

Justification for classification or non-classification

The substance does not meet classification criteria under Regulation (EC) No 1272/2008 for dermal irritation.

The substance does not meet classification criteria under Regulation (EC) No 1272/2008 for eye irritation.

 

For skin irritation, further in vitro skin corrosion testing does not need to be conducted based on the available information allowing a definitive conclusion on the classification of the substance. The substance does not demonstrate significant skin irritation potential necessary for classification and labelling within an available skin irritation in vitro assay (eq. or similar to OECD TG 439) and an available in vivo assay (OECD TG 404).

 

For eye irritation, the weight of evidence indicates that the substance has the potential to cause transient mild irritating effects to the eye but which are insufficient for classification based on the applicants recalculation of the mean scoring and evaluation of the results in three organisms demonstrating that the EU criteria had not been met. Effects in vivo on corneal opacity are non-existent and iritis and conjunctival effects are very low which fully reversed within 72 hours; the overall evidence is indicative of transient and reversible effects on the eye.

 

References:

1. ECHA Guidance on Application on the CLP Criteria, (v5.0, July 2017)